This invention relates generally to the separation of a desired or undesired population or subpopulation from a sample to obtain the desired population or subpopulation alone or an enhanced population or subpopulation with one or more undesired subpopulations removed therefrom. More particularly, the invention is directed to separating the desired or undesired population or subpopulation such as cells from bone marrow or blood, by binding the population or subpopulation to relatively dense particles and utilizing gravity sedimentation to separate the population or subpopulation from the remaining sample supernatant, including a plurality of separation steps.
The enhancement of a population or a subpopulation of a sample such as hematopoietic stem cells (HSC) can be utilized for many types of applications. The sample can be from bone marrow aspirates, vertebral body marrow (VBM), blood (including peripheral, mobilized or cord blood) or other sources from human or other animals, such as rats, mice, dogs, pigs, cows or primates. The term “mobilized” refers to treatment of a patient with appropriate stimulators to enhance the number of HSC cells. Thus, reducing the amount of sample which must be obtained from a patient by bone marrow aspirations or blood pheresis to obtain a sufficient population of HSC. In addition to enhancement of a population or subpopulation, it is often necessary to remove undesired populations or subpopulations. For example, in bone marrow, the removal of all non-Hodgkin's B lymphoma cells can be desired in the case of a B cell lymphoma. If the bone marrow is to be purged of the B cells and reinfused into the patient, it is important that the bone marrow be completely purged and that the bone marrow be not otherwise damaged.
Currently, one approach is to utilize a plurality of magnetic microspheres, typically formed of a polymer based magnetic material of a relatively low density. The microspheres are desired to be of a relatively low density, because the microspheres are mixed with the bone marrow or blood and specifically are designed not to settle out by gravity sedimentation. The microspheres are typically of a small size, generally about or less than one micron in diameter. However, one product sold by Dynal, Inc. of Great Neck, N.Y., utilizes magnetic polymeric microspheres having a nominal diameter of 2.8 or 4.5 microns with a low microsphere density on the order of 1.5 gm/cc. The prior art magnetic microspheres are intended to be maintained in suspension in the sample and consequently are designed for very slow or substantial elimination of gravity settling in the sample suspension.
The magnetic microspheres have at least one antibody bound thereto specific to the population or subpopulation desired to be removed. Often, such as in the Dynal process, a first monoclonal antibody is bound to the cells of interest and a second antibody specific to the first monoclonal antibody is bound to the microspheres. The cells typically are isolated from whole blood or bone marrow and then washed prior to binding the monoclonal antibody thereto, which washing step causes a non-discriminant loss of cells. The microspheres and cells then are mixed together to bind the microspheres to the cells via the first and second antibodies. For purging blood or bone marrow, a sample would be mixed with a plurality of the antibody bound microspheres and then placed in a magnetic field. The remaining sample or supernatant is removed while the microspheres are held in the magnetic field. This procedure typically must be repeated, since a single purging step generally will not deplete a sufficient percentage of the undesired population or subpopulation(s). The goal of purging is to remove all (100%) of the targeted population or subpopulation. This generally is not feasible and the sample is purged as close to one-hundred (100) percent as is feasible.
The magnetic removal procedure presents several problems. The procedure also removes a number of cells non-specifically from other populations during each removal step. This decreases the yield, i.e., the percent of the desired population remaining. A single removal step results in a varying yield of a relatively low percent with each succeeding step also reducing the yield. Further, the magnetic microspheres are relatively expensive.
The magnetic microsphere procedure also has been utilized for enhancing a subpopulation for study of the subpopulation. In this case, the magnetic microspheres are bound to the desired subpopulation and the microspheres with the cells bound thereto are removed from the sample. The subpopulation then can be studied directly or can be removed from the microspheres for study. This procedure is time consuming, on the order of about one (1) to six (6) hours or longer, and arguably does not result in a native subpopulation, since the subpopulation has had at least one monoclonal antibody bound to at least one type of cell antigen.
A further use for purging is the study/enhancement of a specific subpopulation, such as the CD4 population of the lymphocytes (L). In this case the microspheres have monoclonal antibodies bound thereto specific to one or more non-CD4 populations. The removal of the other populations enhances the number of CD4 cells in the total remaining cell population in the sample. However, when the magnetic microspheres are utilized, the nonspecific removal of a portion of the CD4 subpopulation can seriously effect the remaining number of the CD4 subpopulation. The non-specific removal of                cells can become more of a problem when a large sample volume is being utilized, such as five (5) ml and larger, which volume then requires a large number of the magnetic microspheres. When the magnetic microspheres then are placed in a magnetic field, non-specific trapping and removal of other non-targeted cells often occurs.        
Other methods of positive or negative selection, including antibody labeled surfaces, have been used for generating subpopulations of cells from a mixture of different cell types. These methods usually have antibody covalently attached to a plastic surface or to polymer particles in a column. In general, the mixed cell population is combined with the attached antibody, either by adding them to a column and letting them incubate or by letting them settle onto a surface. These procedures work optimally when the red blood cells (RBC's) and plasma have been initially removed from the mixed cell population by preparation of a buffy coat or a mononuclear preparation by density gradients, washing the cells and combining them with the antibody labeled surface. Both methods also require preparation of the separation system and washing with a buffer prior to use, which with incubation times of thirty to sixty (30-60) minutes with the antibody, results in a procedure which takes a minimum of three hours for the column and flask method. These methods can be used for either negative selection or positive selection for the cell population of interest. In both methods, direct separation results in a highly enriched population with resultant loss of non-targeted cells non-specifically. The released cells may have antibody on the cell surface and often are activated by the separation technique, which often is not desirable.
The method and apparatus embodying the invention can be utilized with a variety of immunological reactions, such as immunological reactions involving reactants and cells, virus or biological particles, such as platelets. As utilized herein, cells are defined as animal or plant cells, including cellular bacteria, fungi, which are identifiable separately or in aggregates. Cells are the least structural aggregate of living matter capable of functioning as an independent unit. For example, cells can be human RBC and white blood cell (WBC) populations, cancer or other abnormal cells from tissue, bone marrow and/or from blood samples. Cells suitably tagged or labeled, reasonably can be expected to be operated on by the method and apparatus of the invention in the same manner as the human blood cell or bone marrow examples.
As utilized herein, the term “reactant” defines various molecule(s), such as monoclonal or polyclonal antibodies, which detect and react with one or more specific complementary molecule(s), such as antigens, which are on the surface of a cell. Some examples are given below:
ReactantSpecific MoleculeAntibodyAntigenDrugDrug ReceptorHormoneHormone ReceptorGrowth FactorGrowth Factor ReceptorLectinCarbohydrate MoleculeNucleic AcidComplementary NucleicSequenceAcid SequenceEnzymeCofactor or InhibitorThe reactants couple or bind to the specific molecule(s) on the cells.
It would be desirable to have an effective method of purging or selecting one or more subpopulations without effecting the remaining populations in a sample, such as whole blood or bone marrow. The method should be inexpensive, fast, result in a high yield even after multiple selection steps or rounds and not be restricted in the volume of sample to be acted upon.